Several lines of our research show that the organizational status of regulatory elements in the eukaryotic nucleus can impact strongly on their utilization. We have developed a reagent that will be very useful in testing models related to the domain structure of chromatin and intranuclear organization. We have constructed a chimera between the green fluorescent protein (GFP) and the glucocorticoid receptor (GFP-GR) that functions in cultured cells. GFP-GR will transactivate MMTV reporter constructs as efficiently as wild type GR, and the ligand specificity for transactivation is identical to the untagged receptor. When GFP-GR is expressed in cells, the chimera is initially located in the cytoplasm. Addition of ligand induces complete translocation into the nucleus. When a ligand with full agonist activity is used, GFP-GR will localize within the nucleus on a large number of targets which represent the endogenous chromatin binding sites for GR. When a cell line with a high copy number of integrated MMTV LTR constructs is used, GFP-GR localizes in a highly fluorescent set of foci, which correspond to amplified sets of the MMTV reporter. RU486 is an antagonist of GR that induces DNA binding in vitro, but does not induce transcriptional activation in vivo. The RU486 ligand induces complete nuclear translocation of GFP-GR, but the receptor remains distributed throughout the nucleus with no focal localization. A very important finding was obtained with a cell line, 3134, that harbors a 200-copy, head-to-tail tandem repeat of an MMTV LTR-ras-BPV fusion. Using these cells, a bright ribbon of GFP-GR localization is observed that represents direct binding to the tandem array in living cells. In situ hybridization with MMTV LTR probe shows that this structure corresponds to the tandem array. We have thus detected, for the first time, specific receptor binding to a biologically correct regulatory element in living cells in real time. RU486 gives complete translocation of the receptor into the nucleus, but fails to target GR to the tandem array. This nuclear localization represents a previously undescribed compartment for the glucocorticoid receptor. GR apparently moves through this compartment prior to actual binding to an authentic regulatory element in chromatin.